An ion trap is commonly utilized in a mass spectrometer (MS) as a means for controlling and spatially confining the motions of ions for various purposes. The theory, design and operation of various types of ion traps and associated mass spectrometers are well-known to persons skilled in the art and thus need not be detailed in the present disclosure. One common class of ion traps is the Penning trap, or ion cyclotron resonance (ICR) cell, marketed commercially as a Fourier Transform Mass Spectrometer (FTMS). The Penning trap uses an arrangement of electrodes to apply fixed magnetic and electric fields to confine ions in the radial and axial directions, respectively. An alternating electric field is used to resonantly excite the ions for determination of their mass-to-charge (m/z) ratios. Another common class of ion traps is the Paul or RF (radio frequency) trap, in which alternating electric field gradients are used to confine the ions. The Paul trap may have a three-dimensional (3D) configuration formed by a ring electrode and two opposing end-cap electrodes. An RF trapping field applied to the ring and end caps of the 3D trap confines the ions in both the radial and axial directions. The Paul trap may alternatively have a two-dimensional (2D) configuration formed by a multipole arrangement of parallel electrodes extending in the axial direction and end electrodes positioned at the opposing axial ends of the multipole electrode set. An RF trapping field applied to the multipole electrode set confines the ions in the transverse direction, and DC potentials applied to the end electrodes confine the ions in the axial direction. In Paul traps, the RF trapping field is modulated, or alternatively a supplemental AC resonant excitation field is added, to manipulate the ions for determination of their m/z ratios.
In conjunction with processes such as tandem MS (MS/MS), ion traps may be used to dissociate (fragment) ions into smaller ions to enhance structural elucidation and identification of the molecules that were ionized for investigation. The mechanism for dissociation usually performed in Paul traps is collision-induced dissociation (CID), also referred to as collision-activated dissociation (CAD). CID entails accelerating a parent ion to a high kinetic energy in the presence of a background neutral gas (or collision gas) such as helium, nitrogen or argon. When the excited parent ion collides with the gas molecule, some of the parent ion's kinetic energy is converted into internal (vibrational) energy. If the internal energy is increased high enough, the parent ion will break into one or more product (or fragment) ions, which may then be mass-analyzed. A similar mechanism is employed in Penning traps, known as sustained off-resonance irradiation (SORI) CID, which entails accelerating the ions so as to increase their radius of cyclotron motion in the presence of a collision gas. An alternative to CID and SORI-CID is infrared multiphoton dissociation (IRMPD), which entails using an IR laser to irradiate the parent ions whereby they absorb IR photons until they dissociate into fragments ions. IRMPD is also based on vibrational excitation (VE).
CID and IRMPD are not considered to be optimal techniques for dissociating ions of large molecules such as high molecular-weight or long-chain biopolymers (e.g., peptides, proteins, etc.). For many types of large molecules these VE-based techniques are not able to cause the types of bond cleavages, or a sufficient number of these cleavages, so as to yield a complete structural analysis. Currently, electron capture dissociation (ECD) is being researched as a promising new method for dissociating large molecular ions. In ECD, the well-known technique of electrospray ionization (ESI) is usually selected to form multiply-charged ions of large molecules by proton attachment. The “soft” or “gentle” technique of ESI leaves the multiply-charged ions intact, i.e., not fragmented. The ions are then irradiated by a stream of low-energy electrons. If their energy is low enough, the electrons can be captured by the positively charged sites on the ions. The energy released in the exoergic capture process is released as internal energy in the ion which can then cause bond cleavage and dissociation. Typically, ECD occurs at electron energies less than 3 eV. In addition, “hot” ECD (HECD) may be implemented at higher electron energies (typically 3-13 eV) in which electron excitation precedes capture. With HECD the resulting fragments may undergo secondary fragmentation, which can provide analytical advantages for experiments carried out on many types of molecular ions. For example, HECD allows for distinguishing between isomeric leucine and isoleucine residues. See Zuberev et al., Chemical Physics Letters, 356 (2002) 201-206. For purposes of the present disclosure, the term ECD also encompasses HECD unless specified otherwise.
Thus far, ECD has been investigated mainly in the context of Penning trap-based instruments as the magnetic field facilitates stable control over the electrons. Penning trap-based instruments such as FTMS, however, are not in widespread use because of their high cost and technical complexity. On the other hand, the implementation of ECD in Paul traps and multipole RF storage cells is challenging. These RF-based instruments operate without a magnetic field to provide stability for the electrons. Moreover, due to their use of strong electrical fields, RF instruments deflect electrons and cause electron energies to be increased far above the 20-eV upper limit below which electron capture can take place. At these higher energies, parasitic ion formation by electron impact (EI) results in unwanted ions that contribute to the background signal and additional undesired ion-molecule reactions.
The source of electrons typically proposed for ECD is a device that includes a heated cathode capable of thermionic emission and lenses for guiding the liberated electrons as a beam into the ion trap. This type of device is commonly used in conjunction with EI ionization and other processes requiring the production of an electron beam. However, the simple lens system typically employed in such electron sources does not meet the requirements of ECD. Optimized control of the electron beam for ECD is critical because while high energy levels are needed to remove electrons from the thermionic emitting surface, low energy levels are needed for ECD to successfully occur as noted above. Moreover, a high density of low-energy electrons must reach the region where the target ions are confined to produce a sufficient amount of fragment ions.
More specifically, it is known that the electron flux leaving a heated surface increases with the temperature of the surface. It is further known that intense beams of electrons are subject to a maximum flux that is limited by the space charge associated with electrons in the region of the surface. Due to the space charge, increasing the surface temperature will not further increase the electron flux. The space charge limit of electron flux is related to the potential difference of the emitting surface and the surrounding surfaces. This phenomenon is described by the well-known Child-Langmuir space-charge law in which the current density, J, varies as the 3/2 power of the voltage potential according to the relation J=KV3/2, where K is a known constant. The importance of this is that to form intense beams of electrons from a heated surface it is necessary to employ a large extraction voltage. However, the large extraction voltage produces high energy electrons that are not suitable for ECD. Therefore, means must be employed for slowing down the electrons before they encounter the target ions. An additional problem associated with the formation of intense beams of electrons is the undesired beam divergence that occurs when electrons are decelerated. This can be described by the Law of Helmholz-Lagrange (equivalent to the Abby Sine Law in light optics). A summary of this law is that the product of the lateral magnification, the angular magnification, and the ratio of the final and initial indices of refraction (equal to the square root of the potential for charged particle optics) is equal to unity. This is a statement of Liouville's theorem in statistical mechanics which states that the volume of phase space in non-dissipative systems (collision-free conditions) is conserved. Applying these principles to the context of ECD, consider the case where high accelerating potentials are utilized to produce intense electron fluxes from a heated surface with a beam of small angular divergence. If a simple lens system of two different potentials is utilized to control the electron beam, it follows that a large angular divergence will result when the beam is decelerated in the second lower potential region in an attempt to reduce the electron energy down to the levels required for ECD. If too much spatial spreading of the electron beam is permitted at the location of the target ions, there is no assurance that the electrons will have a low enough energy for ECD and the density (or intensity) of the electron beam at this point may be unacceptably low for producing an abundance of fragment ions. Therefore, a conventional lens system is not effective for appropriately shaping the electron beam so that low-energy electrons can be delivered into the trapping region with the desired properties.
Delivering electrons into an RF trapping region is further complicated by the effect that the RF fields have on the motion and energy of electrons. The RF voltage signal typically applied to the ion trap electrodes to confine the ions has a basic sine wave shape. Thus, over most of the RF cycle the magnitude of the voltage is a relatively large positive or negative value such that the ion trap electrodes will deflect electrons away from their intended path by attraction or repulsion. The sinusoidal waveform provides only a very short window of time, where the signal crosses zero volts, in which an electron beam may be successfully directed into the trap without being perturbed by the RF trapping field. Thus, for many applications it would be better to utilize rectangular impulses or other periodic waveforms that provide longer periods of zero RF voltage during which electrons may enter the trapping region, yet are still effective for trapping ions. Impulse-driven RF trapping for electron capture has been studied extensively by Zerega et al., International Journal of Mass Spectrometry, 132 (1994) 57-65, 67-72, and 135 (1994) 155-164; and by Sadat et al., International Journal of Mass Spectrometry, 107 (1991) 191-203. The latter has proposed the use of a particular form of impulse of the type V(t)=cost(Ωt)/(1-kcost(2Ω), where k=0.5-0.99. A wave form of this type was shown to have a stability region very similar to that of a quadrupole trapping field, but with the advantage that about 50% of time the RF voltage is near zero, thus making it ideal for low energy electron attachment studies.
For many applications, it would also be desirable to increase the internal energy of the target ions so as to change the way fragmentation occurs when the target ions undergo ECD or HECD, and to provide fragment ion information complementary to that obtained by ECD or HECD alone. One way of increasing ion internal energy is to increase ion kinetic energy and allow the accelerated ions to collide with a light collision gas such as helium, similar to the CID techniques described above but without dissociation. However, conventional techniques for increasing ion kinetic energy are difficult to implement in conjunction with ECD. In an RF quadrupole ion trap, ion kinetic energy may be increased by applying a supplemental AC field to an opposing set of electrodes at a frequency that matches the secular frequency of the ion in the trapping field. This means of increasing the kinetic energy thus requires the supplemental field to be in resonance with the ion motion. Moreover, the transverse oscillatory motion also periodically displaces the ions away from the central axis or region where ECD is to occur, and therefore causes the ions to be located at a distribution of electron kinetic energies because this kinetic energy varies in the transverse direction. In a Penning trap, the SORI operation also increases ion kinetic energy. But similar to the resonance condition required by an RF trap for ion excitation, SORI while off-resonance nonetheless requires the use of precise frequencies. Moreover, with SORI the radius of the cyclotron motion of the ion increases and the ions move away from the central axis of the detector cell. It is along the central axis that the low-energy electrons are located due to the effect of the magnetic field. Therefore, while SORI will produce an increase in internal energy due to ion-molecule collisions, the ions will not be located so as to react with the electrons in the trapping cell.
Accordingly, there is a need for apparatus and methods for implementing electron capture dissociation effectively and efficiently in RF confining devices that do not rely on the use of magnetic fields. There is also a need for apparatus and methods capable of selectively implementing either ECD or HECD as desired for a given analysis. There is also a need for apparatus and methods for delivering high fluxes of electrons at very low energies in the ranges required for ECD or HECD to a specific area in an instrument where target ions to be dissociated are trapped. There is also a need for apparatus and methods that provide an electron beam optimized for either ECD or HECD as needed, and optimized for a broad mass range of target ions. There is also a need for apparatus and methods for increasing the internal energy of target ions as a means for enhancing, or modifying the fragmentation pathways provided by, ECD or HECD.